Hepatitis B virus (HBV) is a global major health concern. While Chronic HBV can lead to several severe liver diseases, occult HBV is capable of transmitting infection in hemodialysis units. Since there is lack of information on the pervasiveness of occult HBV in patients regularly receiving hemodialysis in Yemen, this study was designed to fill in this information gap. The study included 150 patients (9 to 75 years), receiving hemodialysis at regular basis. Sera samples were gathered ahead of the hemodialysis session, liver enzymes assessed using commercially available kits, and Hepatitis B surface antigen (HBsAg) as well as other serological markers of HBV measured using commercial enzyme-linked immunosorbent assay (ELISA) kits and rapid cards. HBsAg-negative samples were tested for HBV DNA using SYBR GREEN quantitative polymerase chain reaction (qPCR). All data were statistically analyzed. The prevalence of positive HBsAg was 42.67%, mostly represented by males. No significant relationship existed between the presence of HBsAg and the levels of liver enzymes. Anti-HBs and anti-HBc were detected in 96.5 and 80.2% of patients, respectively. SYBR green qPCR revealed that OBI was detected in ~78% of HBsAg-negative patients which is considered remarkably high. 79% of OBI patients were seropositive whereas only 3% were seronegative. OBI appears to be a significant health issue in hemodialysis patients in Hodeidah city, Yemen. To prevent OBI transmission, the screening of anti-HBc followed by HBV DNA detection in all patients undergoing hemodialysis should be applied. Moreover, hemodialysis patients negative for HBsAg are recommended to be vaccinated for HBV.